Antibiotic Resistance Patterns of Listeria Species Isolated from Broiler Abattoirs in Lusaka, Zambia

L. monocytogenes is a public health threat linked to fast foods such as broiler chickens. This study aimed to verify the occurrence of Listeria species in chickens from abattoirs and evaluate their antimicrobial resistance. In total, 150 broiler carcass swabs distributed as cloacal (n = 60), exterior surface (n = 60), and environmental (n = 30) were collected. Listeria species were characterized using biochemical tests and PCR. We conducted antibiotic resistance tests using the disc diffusion and Etest (Biomerieux, Durham, NC, USA) methods. Overall isolation of Listeria species was 15% (23/150) 95% CI (10.16–22.33), 2% (3/150) 95% CI (0.52–6.19) and 13% (20/150) 95% CI (8.53–20.08) came from environmental swabs and carcass swabs, respectively. Proportions of positive Listeria isolates were L. monocytogenes 74% (17/23), L. welshimeri 22% (5/23), and L. innocua 4% (1/23). Listeria species from the exterior carcass swabs was 61% (14/23), cloacal swabs 26% (6/23), and environmental swabs 3% (3/23). L. monocytogenes had the greatest resistance percentage to the following antibiotics: clindamycin (61%, 10/23), tetracycline 30% (7/23), and erythromycin 13%, (3/23). Isolation of L. monocytogenes in relatively high numbers, including the antimicrobial profiles, suggests a potential risk of the pathogen remaining viable in the food continuum and a public health risk to would-be consumers.


Introduction
Listeria is a facultative anaerobic Gram-positive bacterium with more than 17 recognized Listeria species (spp.) [1]. Listeria has a high distribution in nature, with previous isolation recorded from humans and several animals in the soil, aquatic plants, and assorted types of foods [2]. L. monocytogenes and L. ivanovii are known pathogenic strains affecting humans and animals [3]. From the public health point of view, L. monocytogenes is the most important Listeria spp. that causes a broad spectrum of clinical syndromes, commonly called Listeriosis, with a case fatality of 20-30%. The clinical features range from mild influenza-like illness to meningitis, frequently accompanied by septicemia and meningoencephalitis [3]. Listeria is a ubiquitous pathogen, and the common spp. isolated in food has recorded annual growth rates of about 8% [39], greatly influencing increased poultry consumption due to its perceived economic, safety, and ease of digestion compared to other meat products [40]. Poultry meat sold in smaller pieces that are cheaper encourages consumers to purchase according to what they can afford. In Zambia, Lusaka has increased the demand for chickens, leading to the opening of several chicken abattoirs [41]. With the increase in chicken abattoirs, bacterial contaminants such as Listeria and, more importantly, L. monocytogenes in the production process have become apparent and, thus, a severe public health risk. Despite the high consumption of poultry and the purported risks, there are limited studies on Listeria spp. and their phenotypic antimicrobial resistance, especially in dressed broiler chickens that are among the top meat products readily consumed in the country. This study, therefore, aimed to determine the prevalence of Listeria species isolated from poultry abattoirs located within Lusaka and to determine the resistant patterns of the isolated Listeria spp.

Description and BLAST Analysis of Listeria spp.
To confirm the type of Listeria spp. isolated, BLAST analysis was performed based on the similarity scores ranging from 97% to 100% with reference sequences. Out of the 23 Listeria spp. sequences, 17, 1, and 5 identified as L. monocytogenes, L. innocua, and L. welshimeri, respectively. (Table 2).

Specific Listeria spp. Isolation across Different Abattoirs
When specific abattoir establishment contamination using the total positive samples is considered, abattoir A had the highest contamination of L. monocytogenes with 65% (15/23), while abattoir B reported a higher prevalence of L. welshimeri at 13% (3/23). Meanwhile, abattoir B is the only one that recorded contamination of L. innocua in environmental swabs at a prevalence of 4% (1/23). There was no Listeria spp. isolated from abattoir C ( Table 3).

Distribution of Antimicrobial Resistance Listeria spp. among Abattoirs
Among the antimicrobial resistant isolates of Listeria spp. from abattoirs A and B, the most common resistance phenotype was clindamycin (n = 14/23). The distribution is as follows: L. monocytogenes (n = 10/23) isolated from abattoir A, L. innocua (n = 1/23) isolated from abattoir B, and L. welshimeri (n = 3/23) isolated from abattoir B. Resistance to tetracycline was also recorded in abattoir A (n = 7/23) of which all isolates were L. monocytogenes. In addition, erythromycin resistance was also recorded (n = 5/23) and distributed as follows: L. monocytogenes (n = 3/23) isolated from abattoir A and L. welshimeri (n = 2/23) isolated from abattoir B. While vancomycin showed susceptibility on both the disc and Etest methods, chloramphenicol, gentamicin, and levofloxacin only showed susceptibility on the Etest method (Table 5).

Discussion
This study isolated Listeria spp. from poultry abattoirs located within Lusaka Province and determined A.M.R. Listeria spp. in poultry carcasses. Further, this study found an overall prevalence of 15% (23/150) of Listeria spp. While, the antimicrobials that recorded the highest resistance in Listeria spp. were clindamycin 61% (14/23), tetracycline 30% (7/23), and erythromycin 22% (5/23). The most significant finding of this study is the isolation of L. monocytogenes as the significant contaminant in poultry dressed carcasses, with a prevalence of 74% (17/23). To the best of our knowledge, this study is the first in Zambia to isolate and investigate the antibiotic-resistant patterns of Listeria species isolated from dressed poultry in abattoirs within Lusaka Province. Listeria spp. especially L. monocytogenes, which is responsible for Listeriosis infection, is an important foodborne pathogen [3], and it is known to colonize a variety of food products, including poultry. Several studies have shown that chicken meat is one of the foods involved as a vehicle for L. monocytogenes [42][43][44] infection, ranging from 15.8%, 20%, and 58%, respectively [45][46][47]. Previous reports recorded lower levels of isolation; while the results of this current study were on the higher side than previously reported, isolation variations was due to differences in slaughter processes since the sampled chickens in this study did not come from the same abattoir. Introduction of L. monocytogenes into meat products is possible during processing and, depending on the hygienic measures by food handlers, cross-contamination occurs at this stage [47,48]. Additionally, most chickens sampled in this study were kept under refrigeration temperatures, allowing L. monocytogenes to multiply [48]. In this current study, abattoirs that isolated several Listeria spp. were characterized by improper cleaning of the environment and utensils such as knives. Listeria spp. can form biofilms at refrigeration temperature; thus contributing to the persistence and dissemination of the pathogen [47].
Listeria spp. isolated from two poultry abattoirs using traditional culture and biochemical tests were identified as: L. monocytogenes, L. innocua, and L. welshimeri by sequencing and BLAST analysis of the prs gene Table 2. Although the prs gene does not differentiate the different Listeria spp. on PCR, based on the similarity scores obtained from BLAST analysis results, we infer with some level of confidence, the most likely spp. of Listeria that could have been present in the samples Table 2. This combined approach thus ensured that the isolates under study identified up to spp. level, L. monocytogenes, and L. innocua have been among the common Listeria spp. in foods and processing environments [49,50]. However, the results of this study indicated that L. welshimeri was the second-highest contaminant (22%) ( Table 3). Other authors found different results from this study, with L. monocytogenes and L. innocua being the significant contaminants in most sampled foods and processing plants [42]. These differences could be linked to variances in the sampled foods from the two studies and differences in environmental factors, isolation method, and sample size.
In this current study, L. monocytogenes had the highest prevalence of contamination, 74%, compared to other isolated species of Listeria. It is a severe public health concern, especially if the strains belong to serotypes mainly implicated in Listeriosis and if they are allowed to contaminate other raw foodstuffs due to poor or lack of food safety management systems [28]. Commonly, most Listeriosis cases are reported from high-income countries, while cases are much more likely to go unreported in countries where surveillance systems are lacking. Listeria cases mainly occur sporadically and are reported more in high-income countries, where incidence is relatively low but has a high fatality rate [51]. Several outbreaks of ready-to-eat foods (R.T.E.) have come from processing plants in the past years. For instance, Effimia et al. (2015) reported a 14.4% prevalence of L. monocytogenes in R.T.E. foods products in Greece, while Wu and co-workers observed a 20% prevalence in retail shops in China [51,52]. The world's biggest Listeriosis outbreak happened in South Africa, and it originated from processing plants of R.T.E. foods [53].
Interestingly, in this current study, L. monocytogenes and L. innocua appeared clustered according to specific abattoirs, implying some endemic existence in these specific abattoirs. Equally, different contamination levels were recorded in different abattoirs in this study, with one abattoir recording no Listeria contamination. Listeria spp. contamination is common in processing plants that have compromised hygienic standards, more importantly, if prerequisite programs such as G.M.P.s and Standard Operating Systems (S.O.P.s) are lacking [54]. Listeria spp. are post-processing contaminants indicative of lapses in the entire production system [55]. Evidence shows that poultry carcasses may become contaminated either environmentally during production or from healthy birds in the processing abattoirs [56]. Equally, abattoir designs and equipment used in processing plants significantly influence the quality of food products [57]. A study on poultry abattoirs also found contamination levels that differed from one abattoir [58]. They mainly attributed the differences in the prevalence of contamination to be structurally related to any other factor. These highlighted differences could partly explain why some abattoirs had higher contamination levels than others. Mainly abattoir C did not record any Listeria contamination because they had a functioning food control system against Listeria contamination. As earlier mentioned, Listeria spp. are post-processing contaminants that thrive in environments with poor hygienic processes.
This study revealed lower Listeria spp. contaminations in the environmental swabs compared to carcass swabs. The finding is attributable to the poultry-dressed chicken storing system in the cold rooms. Further, most poultry storage was in cold rooms already packaged without direct contact with the storage environment. They made it impossible for cross-contamination from poultry to the cold-room environment or vice versa. The swabbed environment and food can also influence the isolation of the Listeria spp. One study revealed a significant difference in isolation of Listeria spp. on environmental swabs regarding the type of food processed [59]. This current study has also revealed that carcass contamination does not always mean environmental contamination, such as cold rooms (Table 3).
When the specific part swabbed was considered, a higher prevalence was obtained from exterior swabs indicating higher carcass contamination, possibly because of unhygienic handling practices by food handlers on the processing line. Further, contamination could have arisen from unintended contacts of carcasses with excretions cumulating from the food handlers' skin, mouth, and nose [60,61]. Listeria contamination is suspected to result from cross-contamination of raw processed chicken by improperly cleaned and disinfected processing environments and, to a lesser probability, from the live chickens. The findings of this study concur with similar findings by Cox et al. [62] and Kanarate and others in 2011, who reported processing as a significant hazard of cross-contamination [53]. Comparatively, Ishola and others recorded a higher incidence of 65% on cloacal swabs than on any other parts swabbed [63]. They attributed poultry contaminants to the increased prevalence of contamination with Listeria. These findings were like [64,65], stating Listeria spp. in soil, water, and hay, including bird guts, as the usual habitat for L. monocytogenes [66], including animal feces [67,68].
Clindamycin is widely used in human treatment and animal health to treat Gram-positive bacterial infections [69][70][71]. Antimicrobial resistance in Listeria spp. and L. monocytogenes differs from when the first report of antibiotic-resistant strains was discovered [66]. In this study, L. monocytogenes showed the highest resistance to clindamycin with 61%, and most of these isolates were L. monocytogenes accounting for 43% of this total resistance. Followed by tetracycline 30% resistance with all isolates belonging to L. monocytogenes and erythromycin (21%) distributed as follows: 13%, 8% L. monocytogenes and L. welshimeri, respectively. Both abattoirs A and B reported resistant isolates, as seen in Table 5.
Further, from the results of Table 5, there is an indication that resistance was not clustered according to abattoirs but randomly distributed. The results above also suggest the presence of a high level of resistance to clindamycin, tetracycline, and erythromycin, which are antimicrobial agents classified among the most important in veterinary medicine, especially tetracycline [67]. L. monocytogenes, from the results in Table 5, remains susceptible to the first drugs of choice in the treatment of Listeriosis as prescribed by CLSI guidelines [32]. However, limited studies have recorded high levels of L. monocytogenes resistance to clindamycin, especially from food isolates [68]. Increased clindamycin resistance can influence induction with erythromycin, according to the study by [66]. The mechanism activity of clindamycin is like that of erythromycin, especially in that they share their binding site as recorded by [72]. This clindamycin resistance reported in this current study could partly relate to enzyme inactivation, which is due to possible cross-resistance of the isolation of resistant erythromycin isolates.
This present study also reported erythromycin resistance, contrary to Heelan and others who only reported clindamycin resistance in Listeria spp. [72]. The isolation of erythromycin-resistant isolates in this present study could have partly influenced the higher clindamycin resistance recorded due to the possibility of inducible resistance. Although, further analysis needs to be undertaken for a proper conclusion as this present study did not test for resistant genes to help us ascertain this finding.
Tetracycline reveals the second-highest resistance with a 30% prevalence in L. monocytogenes. Other authors equally reported similar results to the ones conveyed by this study [73,74]. Equally, a systematic review conducted in R.T.E. foods reported tetracycline as the common antibiotic intrinsically resistant to L. monocytogenes classified in the thirdgeneration antibiotics [75]. Furthermore, tetracycline has the most frequently reported resistance phenotype, especially in L. monocytogenes sampled from different sources [70,76]. Although, the major factors that might be contributing to enhanced antibiotic resistance could be complex and may point to many existing lapses. The augmented resistance of tetracycline recorded in this study is because of extensive animal feed additives and veterinary treatment [77]. The need to produce more has boosted the increased use of antibiotics such as tetracycline, especially in poultry [78], which is the cheapest source of protein [37]. Inadequate regulation on antibiotic use, particularly in poultry, can partly influence the abuse of certain drugs [78,79]. The other factor links the farm to a folk concept that should ensure the prudent use of antibiotics in animal foods [80].
Similarly, processing procedures are also essential factors that may foster antibiotic resistance in food during farming practices, slaughtering, and transportation [81]. The passing of time has proved that bacteria can develop resistance mechanisms through the evidence presented by other authors in the last decades. Bacteria such as Listeria can develop mechanisms or acquired resistance through the transmission of genetic materials coming from other bacterial spp., contributing to the alarming numbers of antimicrobial resistance that may cause threats to the successful treatment of various infectious diseases.

Study Design and Site
We conducted a cross-sectional study design in Lusaka, Zambia, from March 2020 to August 2021. Lusaka has four poultry abattoirs, of which only three enrolled because the other abattoir was closed for renovation. Lusaka has the most significant number of poultry abattoirs because of the recent rapid demand for chicken meat. Additionally, the growing population in the capital city has contributed significantly to the speed of transformation seen in this sector to meet production demand. Moreover, the city attracts several production experts, such as personnel who provide training. The convenience of using antibiotics for the treatment of poultry is among the few things that are helping this industry thrive. The coding of three selected abattoirs was with identification letters as Abattoirs A, B, and C. This cohort sampled only included dressed chickens slaughtered at these abattoirs during the study period.

Sample Size and Sampling
We used a circular systematic random sampling method to select a chicken for swabbing [57]. We collected 150 cloacal and exterior surface swabs, including environmental samples. We estimated the sample size on an assumed prevalence of 27.5% [82] at 80% power and a 5% significance level. The abattoir throughputs ranged from 8000 to 20,000 birds per day. We sampled five dressed chickens per batch each day of sampling. At the three abattoirs, we defined a batch as chickens from the same flock and had homogenous characteristics (i.e., same source, same farm). These sampled abattoirs comprised the target population (N) from the sample population (n). We collected a total of 120 carcass swabs and 30 environmental swabs from the poultry abattoirs. A total of 120 carcasses were randomly selected using the circular systematic random sampling system for each batch by selecting five dressed chickens. We selected 120 carcasses in the three abattoirs at the end of the sampling period for bacteriological examination of Listeria and 30 environmental swabs of different batches.

Sample Collection and Processing
We used standard bacteriological sample collection for Listeria contamination on samples collected from three abattoirs [83]. The collection of surface swabs was from the cloacal and exterior parts of the carcasses. A sterile metal template outlined a 5 cm 2 × 5 cm 2 area of parts marked for swabbing, including environmental swabs. The areas outlined by the metal template denoted the parts for swabbing using a sterile moist cotton gauze wrapped around the end of a flat swab stick. Swabbed samples were in screw-cap tubes containing 6 mL Listeria broth [83]. After that, we identified the swabbed samples according to date, ingredient samples (e.g., poultry carcass), batch code, and site name, including comments specific to the sample (e.g., interior or exterior), and were recorded. Identification of samples was by the laboratory code, sampling site/product or ingredient type, date, and site. All samples collected were transported in iceboxes to the microbiology laboratory at the School of the Veterinary Medicine University of Zambia within 3 h of sampling. Upon arrival, we immediately incubated the samples at 37 • C for 48 h. Before isolation from swab diluents, samples were vortexed for 30 s. In environmental and carcass swab samples, they were plated on selective media to detect the target micro-organisms (Listeria) [84].

Isolation and Identification of Listeria spp.
We tested poultry carcass swabs for the presence of Listeria spp. using the standard international methods recommended by the International Organization for Standardization (ISO g11290- 1: 1996, 2004) procedure. First, a 1 g of the sample representative portion from each was inoculated in 9 ml of pre-enriched broth incubated at 37 • C for 24 h, then 1 mL of pre-enriched broth transferred into 9 mL of Fraser broth (Oxoid, Basingstoke, UK) (enriched with Listeria selective supplement) and vortexed for 1 min, followed by incubation at 37 • C for 48 h. A loop-full of the Fraser Broth enrichment culture was inoculated on the Listeria selective agar (Oxoid) surface, incubated at 37 • C for 48 h, and observed for colonies showing typical greenish sheen growth morphology or green-blue colony color of Listeria. The suspected colonies were then sub-cultured onto Nutrient Agar (Oxoid) and later incubated at 37 • C for 24 h to obtain pure colonies. We performed standard biochemical tests on the purified cultures, namely, Gram staining, citrate, urea, indole, motility, oxidase, catalase, and methyl-red tests, to obtain a presumptive diagnosis of Listeria.

Phenotypic Detection of Antimicrobial Resistance in Listeria spp. Isolates
The Kirby-Bauer disc diffusion method was used on Mueller Hinton agar with 5% sheep blood on 23 isolates of Listeria spp. (Oxoid). Penicillin G (10 unit), gentamicin (30 µg), Levofloxacin (30 µg), erythromycin (15 µg), clindamycin (30 µg), trimethoprimsulfamethoxazole (1.25/23.75 µg), (30 µg), chloramphenicol (30 µg), and tetracycline (30 µg) were applied as antibiotic agents. CLSI breakpoints for Listeria spp. only include a few antimicrobial agents such as trimethoprim-sulfamethoxazole, ampicillin, imipenem, and penicillin; therefore, we used CLSI breakpoints for Streptococcus pneumoniae for other antimicrobial agents, and this was because the CLSI guidelines indicated that this was the organism used to interpret breakpoints for L. monocytogenes [32]. The identical 23 isolates and antibiotics had their antimicrobial susceptibility tested using the Etest (Biomerieux) with vancomycin and imipenem because there was no interpretation provided under disc diffusion in the CLSI [32]. The quality control strains included were L. monocytogenes ATCC 19118, Streptococcus pneumoniae ATCC 49619, and Staphylococcus aureus ATCC 25923. We classified the isolates as susceptible, intermediate, or resistant in both methods.

PCR Identification of Listeria
The ZYMO DNA extraction kit was used to extract DNA from presumptive Listeria spp. colonies. For amplification, a partial fragment of the prs gene was targeted on PCR using the primer sequence prs-F (5 -GCT GAA GAG ATT GCG AAA GAA G-3 ) prs-R (5 -CAA AGA AAC CTT GGA TTT GCG G-3 ) we used that amplified a fragment of 370 bp [28]. Briefly, One Taq ® Quick-Load ® from Biolabs and 50 µL reactions of PCR were run. We used a PCR master mix consisting of 25 µL One Taq Quick-load 2X Master Mix with Standard Buffer, 1 uL of 10 µM of each forward and reverse primer, 20 µL of nuclease-free water, and 3 µL of the template. We used thermocycling conditions where initial denaturation was at 94 • C for 1 min, followed by 30 cycles at 94 • C for 45 s, annealing at 53 • C for 45 s, and extension at 72 • C for 2 min, with a final extension step at 72 • C for 5 min. Finally, the expected PCR product band size of 370 bp was visualized on 1.5% agarose gel coated with ethidium bromide.

Purification of PCR Products and Cycle Sequencing
As per the manufacturer's instructions, we utilized a Promega purification kit to purify the PCR products and a sequencing PCR run using brilliant dye terminator V3.1 (Wizard S.V. Gel & PCR Promega clean-up System). The sequencing products were further precipitated using the ethanol precipitation method, followed by denaturation and loading in ABI 3500 Genetic analyzer for sequencing [85].

Sequence Analysis
We first assembled and edited the sequences obtained from this study using the ATGC plug-in in Genetyx ver. 12 genetyx corporation, Tokyo, Japan. The assembled sequences were then subjected to BLAST analysis on the NCBI website (https://blast.ncbi.nlm.nih. gov/Blast.cgi (accessed on 12 February 2022)) to identify the spp. of bacteria isolated and sequenced. All sequences generated in this study were deposited in Japan's DNA Database with accession numbers LC700404 to LC700426.

Conclusions
This study aimed to determine the prevalence of Listeria spp. isolated from poultry abattoirs in Lusaka and determine the resistant patterns of the isolated Listeria spp. The results indicate that L. monocytogenes was the most common isolated spp. Further findings revealed, phenotypically, that clindamycin, tetracycline, and erythromycin had the highest prevalence of antibiotic resistance through the first drugs of choice and remained susceptible. These results are essential for determining poultry safety and treatment of Listerioses by antibiotics. The results can contribute significantly to the prudent use of antibiotics in poultry and possibly prevention of future acquisition of resistance by L. monocytogenes.
Furthermore, this study is the first to have isolated Listeria spp. in poultry carcasses using PCR to amplify the prs gene to confirm the presence of Listeria spp. in Zambia. Equally, there is a need to strengthen the observed phenotypic antibiotic resistance recorded in this current study by screening for resistant genes using PCR methods. No.2020-Jan-006). Permission from Lusaka City Council was equally obtained. We retained confidentiality about specific sampling sites throughout this study.

Informed Consent Statement:
We obtained informed consent from all the abattoirs participating in the study.

Data Availability Statement:
All the sequences from this study were deposited in the DNA Database of Japan with accession numbers LC700404 to LC700426. All the data regarding this manuscript we contained within the text.